In a recent post in “Discount Thoughts” Michael Clarkson reviews a paper examining the dynamics of allostery in PBX1 homeodomain. Another example for a possible conformational selection binding mechanism.

A beautiful post by Michael Clarkson on a beautiful work by himself and friends, published in Nature. It’s always nice when a fellow blogger gets to present his own work. Post is re-blogged as is from “Discount Thoughts”.

Adamantane drugs inhibit the M2 proton channel from influenza A, a tiny tetrameric protein that equalizes pH between the virus and the endosome of the cell that has swallowed it. Over the past few years a great deal of structural evidence has accumulated showing how adamantane drugs work. Problem is that the evidence supports two different models of M2 inhibition. “Discount Thoughts” provides a great review on a mechanism that is truly a tough nut to crack.

The authors used a bioinformatics approach to survey the sequences of bacterial genes, to find clusters of rare codons, so that they could identify patches that would be slow to translate. They found that for proteins longer than about 300 amino acid residues, nearly every transcript contained at least one cluster of slow-translating codons. When the authors used a cell-free E. coli expression system to make some of these proteins and allowed only one round of translation initiation per ribosome, they saw a pattern of translation intermediates that matched the sizes predicted by the location of slow-translating patches.

The authors examined the multi-domain protein SufI. In their prediction of the translation speed, there are four slow spots. Aside from the first one, these appear to correspond to the boundaries of different structural domains in the protein. Experiments with proteases suggested that these domains actually folded during the pauses, as the ribosome-bound translation intermediates were resistant to proteolysis.

Interestingly, when two rare leucine codons were replaced by more common ones (the authors call this SufI D25-28), the whole protein became vulnerable to degradation. Similarly, when extra tRNA for these rare codons was added to the cell-free expression system, the full-length protein became protease-sensitive. This suggests that the slow patches are actually necessary for proper folding of the protein.

It’s often the case that lowering the incubation temperature can improve the expression of certain proteins in E. coli. The authors of this study find that is also true for SufI, as the protease resistance of SufI D25-28 can be restored by lowering the temperature, and thus the overall translation rate. When analogous experiments with SufI D25-28 and tRNA supplementation were carried out in living E. coli, the translocation of SufI into the periplasmic space was reduced by a factor of 10 even though the overall protein concentration was not affected, indicating that the co-translational folding allowed by the rare codons is necessary for proper functioning of the protein in vivo.